The present invention relates generally to a parasite protein, and more particularly to Plasmodium sp. chitinase and uses thereof.
Throughout this application various publications are referenced, many in parenthesis. Full citations for each of these publications are provided at the end of the Detailed Description and throughout the Detailed Description. The disclosures of each of these publications in their entireties are hereby incorporated by reference in this application.
Defining molecular targets for drug or vaccine intervention remains a key strategy for developing new ways to prevent and treat malaria, a disease that exacts an enormous social and economic toll worldwide. A number of investigators have proposed transmission-blocking vaccines as one component of an overall program of malaria control (Kaslow 1997). Such vaccines are designed to induce antibodies in humans that, when ingested by the mosquito along with a Plasmodium-containing blood meal, interfere with the development of the parasite within the mosquito midgut. Animal models of transmission-blocking vaccines, based primarily on two P. falciparum zygote/ookinete surface proteins, Pfs25 and Pfs28, have demonstrated proof of principle (Gozar et al. 1998), but results of human clinical trials have not been reported to date.
A Plasmodium ookinete-secreted enzyme, chitinase (E.C. 3.2.1.14), has been demonstrated to be another target of blocking malaria transmission from humans to mosquitoes (Shahabuddin et al. 1993). Chitinases are found in prokaryotes and eukaryotes (Flach et al. 1992); their biologic roles include cell wall modification (e.g. fungi (Kuranda and Robbins 1991), Entamoebae (Willagomez-Castro et al. 1992) and filaria parasites (Fuhrman and Piessens 1985)), carbon source degradation (e.g. Streptomyces spp. (Ni and Westpheling 1997; Robbins et al. 1988), Serratia marcescens (Roberts and Cabib 1982), and Vibrio spp. (Keyhani and Roseman 1996)), and plant and fungal host defense against chitin-containing pathogens (Flach et al. 1992). One other protozoan pathogen of man, Leishmania donovani, the agent of human visceral leishmaniasis, is known to use a chitinase in its life cycle (Schlein et al. 1991; Shakarian and Dwyer 1998). The Leishmania chitinase is thought to disrupt the sand fly cardiac valve, allowing amastigotes to be regurgitated from the midgut into the skin of the vertebrate host. The Leishmania chitinase is not thought to function in invasion of the arthropod vector per-se (Schlein et al. 1992). In contrast, Plasmodium chitinase is thought to be required for the parasite to invade the mosquito midgut after being taken up in a blood meal (Shahabuddin et al. 1993). Because of its critical biological function in the life cycle of the malaria parasite, the Plasmodium chitinase is a potential target for blocking transmission from the vertebrate host to the mosquito vector (Shahabuddin et al. 1993).
The potential importance of chitinase in malaria parasite biology was first suggested by a transmission electron micrograph showing the P. gallinaceum ookinete penetrating and appearing to focally degrade the chitinous peritrophic matrix (PM) in the Aedes aegypti midgut (Sieber et al. 1991). The PMs of the Plasmodium vectors Anopheles gambiae (which carries human malaria parasites) and A. aegypti (which carries avian malaria parasites) are composed of chitin, a xcex2-1,4-linked polymer of GlcNAc, with intercalated proteins including trypsins and peritrophins (Perrone and Spielman 1988; Shen and Jacobs-Lorena 1997; Shen and Jacobs-Lorena 1998). P. gallinaceum ookinetes secrete active chitinase (Huber et al. 1991; Vinetz and Kaslow 1998). Although chitinases are found throughout the prokaryote and eukaryote kingdoms, the biological function of Plasmodium chitinases must be different, because ookinetes do not contain chitin and there is no evidence that ookinetes use chitin or mono- or oligomers of GlcNAc as a carbon source. Chitinases are critical for allowing the parasite to escape the mosquito midgut, as evidenced by the observation that addition of the chitinase inhibitor allosamidin to a blood meal prevents oocyst development (Shahabuddin et al. 1993). Both P. gallinaceum in A. aegypti and P. falciparum in A. freeborni fail to develop into oocysts in the presence of this inhibitor. This effect could be completely reversed by enzymatic degradation of the peritrophic matris (PM) in vivo, by adding exogenous chitinase to the blood meal. These observations demonstrated that a chitinase is necessary for malaria parasites to invade the mosquito and initiate sporogonic development.
Because of intrinsic biologic interest and the potential for Plasmodium chitinases to be targets of interfering with malaria transmission (for a review of malaria transmission-blocking vaccines and the potential of Plasmodium chitinases as targets, see: Kaslow 1993; Shahabuddin and Kaslow 1993), a need exists for the identification of the malarial parasite chitinase.
To this end, the subject invention provides an isolated nucleic acid molecule encoding a Plasmodium sp. chitinase. The invention also provides an oligonucleotide complementary to at least a portion of the mRNA encoding the Plasmodium sp. chitinase.
The isolated nucleic acid molecules of the invention can be inserted into suitable expression vectors and/or host cells. Expression of the nucleic acid molecules encoding the Plasmodium sp. chitinase results in production of Plasmodium sp. chitinase in a host cell. Expression of the oligonucleotide in a host cell results in decreased expression of the Plasmodium sp. chitinase.
The invention further provides methods of screening a substance for the ability of the substance to modify Plasmodium sp. chitinase function, and a method of obtaining DNA encoding a Plasmodium sp. chitinase.
Further provided is an isolated nucleic acid molecule encoding a Plasmodium sp. chitinase, wherein the nucleic acid molecule encodes a first amino acid sequence having at least 90% amino acid identity to a second amino acid sequence. The second amino acid sequence is as shown in SEQ ID NO:3 or SEQ ID NO:4.
The invention further provides a DNA oligomer capable of hybridizing to a nucleic acid molecule encoding a Plasmodium sp. chitinase. The DNA oligomer can be used in a method of detecting presence of a Plasmodium sp. chitinase in a sample, which method is also provided by the subject invention.
The invention also provides an isolated Plasmodium sp. chitinase, a composition thereof, and antibodies or antibody fragments specific for the Plasmodium sp. chitinase. The antibodies and antibody fragments can be used to detect the presence of the Plasmodium sp. chitinase in samples. The subject invention further provides a method of producing an antibody specific for a Plasmodium sp. chitinase in a host. The method comprises selecting the isolated Plasmodium sp. chitinase or an antigenic portion thereof and introducing the selected Plasmodium sp. chitinase or antigenic portion thereof into a host to induce production of an antibody specific for Plasmodium sp. chitinase in the host. Further provided is an isolated Plasmodium sp. chitinase encoded by a first amino acid sequence having at least 90% amino acid identity to a second amino acid sequence, the second amino acid sequence as shown in SEQ ID NO:3 or SEQ ID NO:4.
The subject invention further provides a method of preventing infection of mosquitoes by Plasmodium sp., the method comprising exposing the Plasmodium sp. to an amount of a compound effective to interfere with function of Plasmodium sp. chitinase, thereby preventing infection of the mosquitoes by the Plasmodium sp.
Further provided is a method of preventing transmission of malaria by a mosquito feeding on a subject that may harbor Plasmodium sp. organisms. The method comprises administering to the subject an amount of a composition of the Plasmodium sp. chitinase effective to induce production of an antibody specific for Plasmodium sp. chitinase in the subject, wherein the antibody inhibits Plasmodium sp. chitinase and is transferred to a mosquito feeding on the subject thereby preventing infection of the mosquito by Plasmodium sp. organisms that may be harbored in the subject.
Alternatively, the method of preventing transmission of malaria by a mosquito feeding on a subject that may harbor Plasmodium sp. organisms can comprise administering to the subject an amount of a compound effective to interfere with function of Plasmodium sp. chitinase in the subject, wherein the compound is transferred to a mosquito feeding on the subject thereby preventing infection of the mosquito by Plasmodium sp. organisms that may be harbored in the subject.
Also provided is a method of preventing transmission of malaria by a mosquito that ingests Plasmodium sp. organisms. The method comprises introducing into the mosquito an amount of a compound effective to interfere with function of Plasmodium sp. chitinase thereby preventing infection of the mosquito by ingested Plasmodium sp. organisms.